MAD1L1 and TSNARE gene polymorphisms are associated with schizophrenia susceptibility in the Han Chinese population

Schizophrenia (SCZ) is a severe mental illness with high heritability. This study aimed to explore the correlation between MAD1L1, TSNARE polymorphisms and SCZ susceptibility. A total of 493 SCZ patients and 493 healthy controls were included. The genotypes of MAD1L1 and TSNARE polymorphisms were identified by Agena MassARRAY platform. Odds ratio (OR) and 95% confidence intervals (CIs) were tested via logistic regression analysis in multiple genetic models and different subgroups. We observed that AG genotype of rs1107592, AG genotype of rs4976976, and CA genotype of rs67756423 decreased the susceptibility to SCZ (p < 0.05). Age stratification analysis showed that the TC genotype of rs12666575, AG genotype of rs1107592, and AG genotype of rs4976976 decreased the risk of SCZ individuals older than 36 years (p < 0.05). In addition, the AG and AA genotype of rs4976976, the CA genotype of rs67756423 were associated with a lower risk of SCZ in males (p < 0.05). In females, the TT genotype of rs12666575 in recessive model, the AG and AA-AG genotype of rs1107592 in heterozygote and dominant model, could reduce the susceptibility to SCZ (p < 0.05). However, no significant association was found after Bonferroni correction. Our results suggest that MAD1L1 and TSNARE genetic polymorphisms exert a protective role in the risk of SCZ. These findings provide evidence that MAD1L1 and TSNARE may serve as potential biomarkers of SCZ. However, a replication experiment in a cohort with large sample size are required to confirm our findings. Trial registration Not applicable.

. Previous genetic studies have identified some candidate genes (AKT1, 5-HTT, COMT) as risk genes for SCZ to illustrate the biological mechanism of this disorder [5]. However, the exact roles of these candidate genes in SCZ pathogenesis were not fully established.
Mitotic arrest deficient-like 1 (MAD1L1) is a component of the mitotic spindle-assembly checkpoint which prevents the onset of anaphase until all the chromosomes are properly aligned at the metaphase plate [7]. MAD1L1 involved in tumor suppression and cell cycle control. A large body of literature has demonstrated that the expression of MAD1L1 is abnormal in breast cancer, smallcell lung cancer, and other cancers [8,9]. Besides, it was found that MAD1L1 was related to the reward systems functioning in healthy adults [10]. In a recent study found that MAD1L1 antigene showed increased IgG level in SCZ patients compared with control subjects [11]. Zhao et al. indicated that single nucleotide polymorphism (SNP) in MAD1L1 was significantly associated with bipolar disorder in Chinese people [12]. Nevertheless, there are few studies on the role of MAD1L1 variants in SCZ development.
T-SNARE domain-containing 1 gene (TSNARE1) may have evolved from the harbinger transposon superfamily within the vertebrate lineage [13]. It has been suggested that TSNARE possesses functions related to transcriptional regulation, nuclear import, and DNA binding [14]. Then, bioinformatic predictions indicated it may bind SNARE and have SNAP receptor activity. Additionally, a genome-wide association study (GWAS) meta-analysis has reported that TSNARE1 rs10098073 and rs4129585 were closely related to SCZ and bipolar susceptibility in Caucasians [15]. This was in line with the discovery of Gu et al., which indicated a significant correlation between rs10098073, rs4129585 in TSNARE and SCZ risk in Southeast Chinese Han and Zhuang people [16]. However, the relationship between other SNPs polymorphisms in TSNARE and SCZ susceptibility has not been explored in the Northwest Chinese Han population.
In the present study, we mainly focused on the role of MAD1L1 and TSNARE1 in the pathogenesis of SCZ. We evaluated the association of MAD1L1 rs10275045, rs12666575, rs1107592 and TSNARE1 rs4976976, rs67756423 with SCZ risk in the Northwest Chinese Han population. These findings will provide insights into the pathogenesis and development of SCZ.

Study subjects
A total of 986 individuals, which included 493 SCZ patients and 493 controls, were enrolled from Xi'an Mental Health Center. Schizophrenia was identified by two psychiatrists on the basis of the Tenth Revision of International Classification of Diseases. Patients who met the following conditions were excluded: (1) mental diseases induced by organic brain syndrome, (2) neurological diseases, (3) mental retardation, (4) severe brain injury, (5) non-cooperating patients with superexcitation, (6) pregnant or breastfeeding women. Inclusion criteria for healthy controls were individuals without family history of mental disorder, severe head injury, febrile convulsion in childhood or infant stage. Moreover, we used G*power software to calculate the minimal required sample size based on the probability of a typeIerror of alpha = 5%, typeIIerror of beta = 15% (power = 85%), effect size of 0.2. This calculation yielded a sample consisting of at least 450 cases and 450 controls. Then, we recruited 493 cases and 493 controls in this study.
The legal guardian of these participants provided informed consent documents on their behalf. This study got approval of the Ethics Committee of Xi'an Mental Health Center and followed the Declaration of Helsinki.

SNP genotyping
Peripheral blood samples were collected from each subject. DNA was isolated from venous blood sample by the GoldMag DNA purification kit (GoldMag Co. Ltd, Xi′an, China) in accordance with the user's protocol, then quantified by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). The SNPs in the MAD1L1, TSNARE1 genes were chosen based on the minor allele frequency (MAF) > 0.05 in Han Chinese from the 1000 Genome Projects. Three SNPs (rs10275045, rs12666575, rs1107592) in MAD1L1 and two SNPs (rs4976976, rs67756423) in TSNARE1 were selected in the present study.
Primers of the five SNPs are listed in Additional file 1: Table S1. PCR reactions were performed in a buffer containing 1 μl DNA, 0.5 μl PCR Buffer, 0.4 μl MgCl 2 , 0.1 μl dNTP Mix, 1.0 μl primer mix, and 0.2 μl Taq ligase in a final reaction volume of 5 μl. The reaction mixture was

Data analysis
We performed Pearson's χ 2 test and student's t-test to assess the differences in gender and age of study populations, respectively. Hardy-Weinberg equilibrium (HWE) was examined by Pearson's χ 2 test. The distribution of SNP allele and genotype between SCZ patients and healthy controls were tested by χ 2 test. Odds ratio (OR) and 95% confidence intervals (CI) were applied to estimate the relationship between MAD1L1, TSNARE1 gene and SCZ risk by logistic regression analysis in multiple inheritance models and subgroup. We also evaluated the SNP-SNP interaction in the risk of SCZ using multifactor dimensionality reduction (MDR). Statistical power and false positive report probability (FPRP) values were calculated by the Excel spreadsheet which was offered on Wacholder's website [17]. The functional role of these SNPs was predicted by HaploReg database (https:// pubs. broad insti tute. org/ mamma ls/ haplo reg/ haplo reg. php). The differences were deemed significant at p < 0.05, whereas a value of corrected p < 0.05/5 was considered significant after Bonferroni correction.

Study subjects
Totally, 493 patients (261 men and 236 women) of SCZ and 493 healthy controls (257 men and 232 women) were enrolled with a mean age of 36.47 ± 13.20 years and 36.50 ± 11.89 years, respectively (Table 1). There were no statistical differences in age (p = 0.968) and gender (p = 0.799) between the two groups.

Basic information for the candidate SNPs
Three SNPs (rs10275045, rs12666575, rs1107592) in MAD1L1 and two SNPs (rs4976976, rs67756423) in TSNARE1 were successfully genotyped. In Table 2, we described the details of the selected SNPs regarding SNP ID, gene, chromosomal position, role, MAF. All SNPs were following HWE (p > 0.05) and were found in the intron region.
Therefore, the best model was the three-locus model, a combination of rs10275045, rs1107592, and rs4976976, with the highest testing accuracy and perfect CVC.

FPRP analysis
FPRP and statistical power were calculated for all positive results. As shown in Table 6, at the prior probability of 0.25 and FPRP threshold of 0.2, all significant results of MAD1L1 and TRNARE polymorphisms remained noteworthy.

Discussion
In this case-control study, our results found that rs1107592 in MAD1L1, rs4976976, and rs67756423 in TSNARE were related to a decreased risk of SCZ in the overall analysis. In addition, MAD1L1-rs12666575, -rs1107592, and TSNARE-rs4976976 significantly decreased the occurrence of SCZ individuals aged > 36 years. Subsequently, the stratification results based on age were shown that TSNARE-rs4976976, -rs67756423 in males and MAD1L1-rs1107592,-rs12666575 in females are associated with a lower risk of SCZ. These results suggested that MAD1L1 and TSNARE genetic polymorphisms were associated with SCZ susceptibility and played a protective role in the development of SCZ. MAD1L1 is located at human chromosome 7q22.3 and involves cell cycle control and tumor suppression. Recently, some research has reported that MAD1L1 rs12666575 was related to SCZ risk in different genetic backgrounds. For example, Sleiman et al. demonstrated that rs12666575 was associated with SCZ susceptibility in a mixed-ancestry cohorts from Caucasians, African Americans, and Asians in 2013 [15]. A genomewide association study also discovered that rs12666575 reduced the incidence of SCZ in the Swedish sample [18].   This was consistent with our results, which found the TC and TT genotype of rs12666575 could decrease the risk of SCZ in different subgroups (age > 36 years old and women, respectively). Rs10275045 is located in the intron region of MAD1L1. A study showed that rs10275045 was associated with SCZ risk in European ancestry [19]. However, the relationship between rs10275045 and SCZ susceptibility was not observed in the Chinese Han population. One possible reason for the contradiction is the genetic heterogeneity of SCZ in individuals of different ethnic groups. Besides, our results revealed that the AG phenotype of rs1107592 was associated with a lower incidence of SCZ in the overall. Stratification analysis also showed that rs1107592 decreased the susceptibility to SCZ subjects with age > 36 years. And the AG and AA-AG genotype of rs1107592 played a protective role in SCZ risk of females. To the best of our knowledge, a meta-analysis study has indicated that rs1107592 was related to the susceptibility to SCZ, but OR values were not reported [16]. These results demonstrated that MAD1L1 polymorphisms involved in the occurrence of SCZ and exerted a protective role in SCZ. TSNARE is located at human chromosome 8q24.3 and may have a function in intracellular protein transport and synaptic vesicle exocytosis. Recently, the role of TSNARE in SCZ has attracted the attention of researchers. For example, previous analyses showed that TSNARE rs10098073 and rs4129585 were related to SCZ susceptibility in Caucasians [15]. Similarly, Gu et al. indicated a significant correlation between rs10098073, rs4129585 in TSNARE and SCZ risk in Southeast Chinese Han and Zhuang people [16]. However, there were few studies about TSNARE other polymorphisms and SCZ susceptibility. In the present study, our results discovered that the AG phenotype of rs4976976 and the CA genotype of rs67756423 were related to a lower risk of SCZ in the overall. Then, we further stratified analysis showed that the AG and AA genotype of rs4976976, the CA genotype of rs67756423 decreased the susceptibility to SCZ in males. Additionally, the AG phenotype of rs4976976 reduced the risk of SCZ with age > 36 years in the heterozygote model. These results indicated that TSNARE polymorphisms are associated with susceptibility to SCZ.
In this study, rs10275045, rs12666575, rs1107592, rs4976976 and rs67756423, located in the intron region of MAD1L1 and TSNARE1, might be associated with the regulation of motifs changed, NHGRI/EBI GWAS hits, GRASP QTL Hits, selected eQTL hits, enhancer histone marks and DNAse, suggesting their potential function in SCZ. In addition, some studies provided evidence to support that intronic SNPs alter the susceptibility to diseases by regulating gene expression [20,21]. Therefore, we speculated that MAD1L1 and TSNARE polymorphisms may affect the MAD1L1 and TSNARE expression to alter Table 6 False positive report probability of the association MAD1L1 and TRNARE polymorphisms and SCZ susceptibility SCZ, schizophrenia; OR: odds ratio; CI, confidence interval p a < 0.05 indicates statistical significance b The level of false positive report probability threshold was set at 0. 2